The goal of the research efforts in the Retrovirus Assembly Section is to extend our understanding of basic mechanisms in retroviral replication and pathogenesis. This understanding may lead to new methods of combatting retrovirus-induced disease, including AIDS. There appear to be several different modes of interaction between retroviral proteins and nucleic acids, each with important functional consequences for viral replication. First, an exquisitely specific recognition by the Gag polyprotein (the structural protein of the virus particle) selects the viral RNA for packaging during virus assembly. This recognition involves zinc fingers in the protein. We are studying the mechanism by which the Gag protein recognizes and packages the genomic RNA of the virus during assembly in vivo. Our research strongly suggests that the recognition signal involves the three-dimensional structure formed by a dimer of genomic RNA molecules. We are studying the structure of the dimer linkage and its possible role in packaging of viral RNA. Our mutational studies also show that the zinc fingers have other crucial functions, in addition to their role in the recognition process. These additional functions are now under investigation. Second, the Gag polyprotein and its cleavage product, the nucleocapsid (NC) protein, exhibit nucleic acid chaperone activity. That is, they transiently destabilize base pairs, catalyzing conformational transitions to the optimally base-paired structure in a nucleic acid molecule. This sequence-independent activity is used before or during virus assembly, when the Gag polyprotein promotes the annealing of a cellular tRNA molecule to the viral RNA; the tRNA is the primer for reverse transcription when the virus infects a new host cell. The activity is used again during virus maturation (i.e., after Gag is cleaved by the viral protease), when NC induces a conformational rearrangement in the viral RNA dimer within the particle. The activity also appears to be crucial during reverse transcription, facilitating both polymerization and strand-transfer steps during proviral DNA synthesis; recent data suggest that it may be important during the integration of the DNA into the host chromosome as well. We are studying the molecular mechanism underlying the nucleic acid chaperone activity of these proteins. Third, the Gag polyprotein interacts with either the viral RNA or, alternatively, cellular mRNA molecules, using them as "scaffolding" in the assembly of virus particles. The presence of cellular mRNA molecules in particles lacking the viral RNA was particularly obvious when the virus-producing cells contained an alphaviral vector. This is because alphaviral vectors replicate their RNAs to extraordinary levels, resulting in a nearly monodisperse population of mRNAs in the cell; under these conditions the alphavirus-derived mRNA replacing retroviral RNA was easy to detect in retroviral particles. We have also found that the HIV-1 Gag polyprotein is able to assemble (in the presence of nucleic acid) into minute spherical virus-like particles in a completely defined system in vitro. The nucleic acid requirement can be fulfilled by oligodeoxynucleotides as short as 10-15 nucleotides. The virus-like particles are only 25-30 nm in diameter, whereas the cores of authentic virions formed in mammalian cells are ~100 nm in diameter. We found that if assembly reactions are performed in the presence of reticulocyte lysates, particles of 100 nm, rather than 25-30 nm, are formed. Therefore, mammalian cells contain a factor that alters the radius of curvature with which Gag polyprotein molecules interact with each other during the assembly process. We have now identified this factor as inositol pentakisphosphate (IP5). We are now analyzing the molecular mechanism of this effect of IP5 on the interactions between HIV-1 Gag molecules. We are also attempting to demonstrate that IP5 or related compounds contribute to the correct assembly of HIV-1 virus particles in vivo. In all retroviruses, the Gag polyprotein is targeted to the plasma membrane of the virus-producing cell. While we have some understanding of how these proteins bind to membranes, the reason for the specificity for the plasma membrane is completely unknown. We are also exploring the possibility that phosphatidylinositides are involved in targeting these proteins to the plasma membrane. Many of these experiments are using confocal microscopy. In addition, we have used surface plasmon resonance technology to analyze the binding of HIV-1 NC protein to very short oligonucleotides. Although NC is probably capable of binding to any single-stranded DNA or RNA, these studies showed that it exhibits profound sequence preferences. We are engaged in a detailed investigation of the binding of NC and Gag to short, well-defined oligonucleotides; this information should help us understand the various interactions with nucleic acids discussed above (i.e., chaperone activity of both NC and Gag, assembly of virus-like particles by Gag, and the exquisitely specific encapsidation of genomic RNA by Gag during virus assembly in vivo). The existence of convenient in vitro assays for specific nucleic acid binding by NC and for virus assembly by Gag has made it possible to screen large libraries of compounds for inhibitors of these functions. Compounds capable of interfering with these functions have been identified and are now being assayed for their ability to prevent HIV-1 replication in cultured cells.